ISOLATION AND IDENTIFICATION OF STREPTOMYCES SPECIES WITH ANTIFUNGAL ACTIVITY AGAINST FUNGAL PHYTOPATHOGENS CAUSING PAWPAW ROT

ABSTRACT

The present study isolated and identified Streptomyces species from soil samples having antifungal activity against selected fungal phytopathogens (Aspergillus niger, Rhizopus stolonifer, Fusarium solani, Aspergillus flavus). Soil samples were collected from different locations of Michael Okpara University of Agriculture, Umudike (MOUAU). The 10 soil samples were cultured by spread plate inoculation method on Nutrient Agar, Sabouraud Dextrose Agar and Tryptone Soya Agar media. Totally 13 Streptomyces  species were isolated and tested for antifungal activity by agar well diffusion method against the fungal phytopathogenic  microorganisms. Isolate IS12, IS51, IS72, and IS91 were moderately to highly active, while IS51 exhibited the highest antifungal activity against Aspergillus niger (19mm) and Aspergillus flavus (18mm) and IS72  showed the highest activity against Rhizopus stolonifer (19mm) and  Fusarium solani (16mm). The zone of inhibition for crude extracts were also determined by agar well diffusion method. These isolates had antifungal activity and could be used in the development of novel antibiotics for agricultural purposes or employed as biocontrol agents to controlling plant (pawpaw) fungal diseases. Soil samples could be an interesting source to explore for antifungal secondary metabolites and there is not any scientific report on the isolation of Streptomyces species producing antifungal metabolites from the areas of study.

CHAPTER ONE

            1.0           INTRODUCTION    

Streptomyces is the well-known genus of Actinomycetes which is represented in nature by the largest number of species and varieties. Soil is the main source of Actinomycetes (Takahashi and Omura, 2010). Although this group of bacteria can be found in aquatic habitats, they are mainly transient in nature (Kowato and Shinobu, 2013). From the beginning of the 20^th century, the genus Streptomyces has become very impotent for the production of antibiotics in controlling human ailment as well as animal and plant diseases specially crop diseases. (Williams et al, 2010). Almost all  Streptomyces  species have been proved to be antibiotic producers (Aghighi et al; 2010), From economic and medical view points, extensive researches have been carried out worldwide to screen antibiotic producer Streptomyces. The intense screening of soil for Streptomyces spp. from the 1940s onward led to the discovery of many new chemical compounds including antibiotics and other bioactive substances (Antonieta et al; 2012). As interest in a particular product intensified so did the interest in the identity of the producing microorganisms. Since the late 1980s the search for new antibiotics has been downsized in many companies to accommodate interests in non-infectious diseases. Nonetheless, products from Streptomyces Species are being evaluated in new detection systems (Hucker and Conn, 2013). However, researchers in this field assume that only 10% of the total microbial population of the earth has been isolated and characterized. So still there is opportunity to isolate and identify new Streptomyces  species and  new compounds from this genus (Williams et al, 2010). In an attempt to do these, different researchers considered different characters such as morphology, actual criteria such as growth on the different media, biochemical characters like utilization of carbon sources  etc. However, there is a big gap between traditional approaches and modern methods for the isolation, classification, and identification of Streptomycetes (Antonieta et a; 2012).

1.1          SOIL MICROORGANISMS

Soil microorganisms provide an excellent resource for the isolation and identification of the therapeutically, agriculturally and industrially important products. Among them, Actinomycetales are an important group (Berdy, 2005). The order Actinomycetales is composed of approximately 80 genera, nearly all from terrestrial soils, where they live primarily as saprophytes, water and colonizing plants showing marked chemical and Morphological diversity, but from a distinct evolutionary line (Kavitha et al; 2010). Actnomycetes are Gram-positive bacteria with high guanine + cytosine (GC) content of over 55%  (Vetsigian and Roy,  2011), in their DNA (Deoxyribonucleic acid), which have been recognized as sources of several secondary metabolites, antibiotics, and bioactive compounds that affect microbial growth (Ikeda et al; 2013). Actnomycetes have filamentous nature, branching pattern, and conidia formation, which are similar to those of fungi. For this reason, they are also known as ray fungi (Wang et al; 2010). Actnomycetes produce branching mycelium which may be of two types, viz; substrate mycelium and acrial mycelium. Streptomyces are the dominant of all actinomycetes (Okami and Okazaki, 2010). A large number of actinomycetes have been isolated and screened from soil in the past several decades, accounting for 70 – 80% of relevant  secondary metabolites available commercially (Watve et al; 2010). Actinomycetes are potential sources of many bioactive compounds (Pallavi et al; 2013), which have diverse clinical effects, and important applications in agriculture, industry and in human medicine (Watve et al; 2010). It has been estimated that approximately one third of the thousands of naturally occurring antibiotics have been obtained from actinomycetes (Garza et al; 2012). Actinomycetes derived from marine and coastal  habitats continue to provide pharmacologically important secondary metabolites and considered as an ongoing source of unique and novel chemical structures (Subbarao, 2012) Especially, Streptomyces  are renowned sources of novel secondary metabolites which have a range of biological activities such as antimicrobial, anticancer, and immunosuppressive activities (Kim et al; 2014). Such Streptomyces  are continuously explored for antimicrobial drug discovery (Dehnad et al; 2010).  Production of secondary metabolites by microorganisms highly depends on the strains and species of microorganism and their nutritional and cultural conditions (Wang et al; 2010). Soil is the major reservoir of microorganisms that produce antibiotics. Considering that soil is densely packed with microorganisms, it is not a wonder that many bacterial and fungal species have evolved over the cons to develop ways of inhibiting their neighbours for the benefit of their own growth (Kumar et al; 2010) an antibiotic made by a microbe can inhibit many other soil microbes (Fall et al; 2011). The bacteria genera Bacillus and Streptomyces  along with fungal genera Penicillium and Cephalosporium are commonly found in soil. The genus Streptomyces are the most prolific antibiotic producers and although bacteria, are a unique subgroup of bacteria called the actinomycetes. Although soil has historically been used to find new antibiotic producers, at present, many of the old antibiotics  are now been manipulated in the laboratory and chemically modified to form new versions of older antibiotics (Fall et al; 2011). Soil may be regarded as a three phase system composed of solids, liquids and gases, dispersed to form a heterogenous matrix. On the whole, the soil is composed of five major components, these include mineral matter, water, organic matter, air and living organisms (Sujatha et al; 2013). The various components of the soil environment constantly changes and the quantity of these constituents are not the same in all soil but vary with locality. Living portion of the soil body include small animals, and microorganisms but it is generally considered that its microorganisms plays the most important role in the release of nutrient and carbondioxide for plant growth (Kumar et al; 2010). The number and type of bacteria present in a particular soil would be greatly influenced by geographical  location such as soil temperature, soil type, soil pH, organic matter content, cultivation,  aeration and moisture content (Selvakumar et al; 2010). The aim of the present study was to isolate and identify Streptomyces species from soil in Michael Okpara University of Agriculture, Umudike,  Abia State  that would produce antifungal effect against some selected fungal phytopathogens causing rot in  pawpaw.

1.2                OBJECTIVES

(1)    To isolate and identify antibiotic producing Streptomyces species from the soil within Michael Okpara University of Agriculture, Umudike, Abia State.

(2)  To evaluate its antifungal activity against pawpaw rot fungal phytopathogens.

(3)     Extraction and antifungal sensitivity testing of crude extracts produced by potential isolates.

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